Optical glucose sensor chip and method of manufacturing the same

ABSTRACT

An optical glucose sensor chip includes a substrate, a pair of optical elements formed on a surface of the substrate for introducing light into the substrate and for emitting the light from the substrate, and a glucose sensing membrane formed on the surface of the substrate at a position between the optical elements. The sensing membrane includes a color reagent substrate, a first enzyme which oxidizes or reduces glucose, a second enzyme that generates a material which makes the color reagent substrate exhibit color by a reaction with a product obtained by oxidation or reduction of glucose, a nonionic cellulose derivative, and an ionic polymer into which a buffer is incorporated. At least one of the first and second enzymes is coated with the ionic polymer, and the color reagent substrate. The first and second enzymes, the buffer and the ionic polymer are supported by the nonionic cellulose derivative.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromprior Japanese Patent Application No. 2006-198173, filed Jul. 20, 2006,the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an optical glucose sensor chip and amethod of manufacturing the optical glucose sensor chip.

2. Description of the Related Art

As an optical glucose sensor chip, for example, a less invasive typeblood sugar level measuring chip has been developed which indirectlymeasures a blood sugar level by extracting body fluid of subcutaneoustissues. This sensor chip has a structure including a glass substrate, apair of gratings which are formed on a surface of the substrate andintroduce light to or emit the light from the substrate, and a glucosesensing membrane which is positioned between the gratings and formed onthe surface of the substrate. This glucose sensing membrane contains acolor reagent substrate (for example, 3,3′5,5′-tetramethylbenzidine[TMBZ]), a first enzyme (for example, glucose oxidase [GOD]) thatoxidizes or reduces glucose, a second enzyme (for example, peroxidase[POD]) generating a material that reacts with a product obtained by theoxidation or reduction of glucose to make the color reagent substrateexhibit color, and a film forming high-molecular compound (for example,a cellulose derivative such as carboxymethyl cellulose [CMC]).

When a sheet gel is disposed between the skin and the above sensingmembrane to apply an electric field in a glucose sensor chip having sucha structure, glucose in a subcutaneous tissue solution penetrates thegel from the skin and reaches the above sensing membrane. At this time,the above TMBZ as a color reagent substrate in the sensing membranemakes to exhibit color due to the reaction between glucose and GOD orPOD. When light is made to be incident to the above substrate and to berefracted on the surface of the substrate and on one of the abovegratings, the light propagates through an interface between the abovesubstrate and the sensing membrane containing color-formed TMBZ, isrefracted on the interface between the substrate and the other gratingand is received by, for example, a light receiving element. Theintensity of the received laser light is less than the intensity(initial intensity) of the laser light received by the light receivingelement when the above sensing membrane has not exhibited color. Forthis reason, the concentration of the above glucose can be detected fromthe ratio of reduction of the above light intensity.

However, when the above sensing membrane is stored and used for a longtime, the activity of the first and second enzymes in the membranerapidly deteriorates. Examples of the cause of the deterioration includea variation in the pH of the sensing membrane, a variation in the ionicstrengths of the first and second enzymes and hydrolysis of the firstand second enzymes. When the first and second enzymes deteriorate, thefirst enzyme reacts insufficiently with glucose which is the subject tobe measured. The reduction in the reactivity with glucose reduces thegeneration of the material that aids the color-forming material obtainedby the subsequent reaction with the second enzyme in exhibiting color,with the result that this causes a reduction in the reactivity with thecolor-forming material and a reduction in the degree of the color to beexhibited, bringing about a deterioration in the sensitivity of theglucose sensor chip.

BRIEF SUMMARY OF THE INVENTION

The present invention is to provide an optical glucose sensor chip whichcan limit or prevent the deterioration of the first and second enzymesin the sensing membrane with time and to provide a method ofmanufacturing the optical glucose sensor chip.

According to a first aspect of the present invention, there is providedan optical glucose sensor chip comprising:

a substrate;

a pair of optical elements which formed on a principal surface of thesubstrate for introducing light into the substrate and for emitting thelight from the substrate; and

a glucose sensing membrane formed on the principal surface of thesubstrate at a position between the optical elements;

wherein the sensing membrane includes a color reagent substrate, a firstenzyme which oxidizes or reduces glucose, a second enzyme that generatesa material which makes the color reagent substrate exhibit color by areaction with a product obtained by oxidation or reduction of glucose, anonionic cellulose derivative, and an ionic polymer into which a bufferis incorporated,

at least one of the first and second enzymes is coated with the ionicpolymer, and

the color reagent substrate, the first and second enzymes, the bufferand the ionic polymer are supported by the nonionic cellulosederivative.

According to a second aspect of the present invention, there is providedan optical glucose sensor chip comprising:

a glass substrate;

a pair of optical elements formed on a principal surface of the glasssubstrate for introducing light into the glass substrate and foremitting the light from the glass substrate;

a light-reflecting path layer formed on the principal surface of thesubstrate on which the optical elements are formed and made of a resinhaving a higher refractive index than the substrate; and

a glucose sensing membrane formed on the light-reflecting path layer ata position between the optical elements;

wherein the sensing membrane includes a color reagent substrate, a firstenzyme which oxidizes or reduces glucose, a second enzyme that generatesa material which makes the color reagent substrate exhibit color by areaction with a product obtained by oxidation or reduction of glucose, anonionic cellulose derivative, and an ionic polymer into which a bufferis incorporated,

at least one of the first and second enzymes is coated with the ionicpolymer, and

the color reagent substrate, the first and second enzymes, the bufferand the ionic polymer are supported by the nonionic cellulosederivative.

According to a third aspect of the present invention, there is provideda method of manufacturing an optical glucose sensor chip, comprising:

preparing a glucose sensing membrane-forming coating solution by usingany of: (a) a method in which at least one of a first enzyme thatoxidizes or reduces glucose and a second enzyme that generates amaterial which makes a color reagent substrate exhibit color by areaction with a product obtained by oxidation or reduction of glucose ismixed in advance with an aqueous solution containing an ionic polymerand a buffer, and the mixed solution is added to and mixed with theother enzyme, a color reagent substrate and a nonionic cellulosederivative; (b) a method in which the first and second enzymes arerespectively mixed in advance with an aqueous solution containing anionic polymer and a buffer, and the respective prepared mixed solutionsare added to and mixed with a color reagent substrate and a nonioniccellulose derivative; or (c) a method in which both the first and secondenzymes are mixed in advance with an aqueous solution containing anionic polymer and a buffer, and the prepared mixed solution is added toand mixed with a color reagent substrate and a nonionic cellulosederivative;

forming a pair of optical elements on a substrate for introducing lightinto the substrate and for emitting the light from the substrate; and

applying the glucose sensing membrane-forming coating solution to asubstrate area positioned between the optical elements, followed bydrying to form a glucose sensing membrane.

According to a fourth aspect of the present invention, there is provideda method of manufacturing an optical glucose sensor chip, comprising:

preparing a glucose sensing membrane-forming coating solution by usingany of: (a) a method in which at least one of a first enzyme thatoxidizes or reduces glucose and a second enzyme that generates amaterial which makes a color reagent substrate develop color by areaction with a product obtained by oxidation or reduction of glucose ismixed in advance with an aqueous solution containing an ionic polymerand a buffer, and the mixed solution is added to and mixed with theother enzyme, a color reagent substrate and a nonionic cellulosederivative; (b) a method in which the first and second enzymes arerespectively mixed in advance with an aqueous solution containing anionic polymer and a buffer, and the respective prepared mixed solutionsare added to and mixed with a color reagent substrate and a nonioniccellulose derivative; or (c) a method in which both the first and secondenzymes are mixed in advance with an aqueous solution containing anionic polymer and a buffer, and the prepared mixed solution is added toand mixed with a color reagent substrate and a nonionic cellulosederivative;

forming a pair of gratings on a substrate for introducing light into thesubstrate and for emitting the light from the substrate;

forming a light-reflecting path layer made of a resin having a higherrefractive index than the substrate on a principal surface of thesubstrate on which the optical elements are formed; and

applying the glucose sensing membrane-forming coating solution to thepart of the light-reflecting path layer positioned between the opticalelements, followed by drying to form a glucose sensing membrane.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a sectional view showing a glucose sensor chip according to afirst embodiment;

FIG. 2 is a sectional view showing a glucose sensor chip according to asecond embodiment; and

FIG. 3 is a view showing a variation in the absorbance (sensitivity) ofa glucose sensor chip of each of Examples 1 and 2 and ComparativeExample 1 with the passage of a storage time.

DETAILED DESCRIPTION OF THE INVENTION

An optical glucose sensor chip according to an embodiment of the presentinvention will be explained in detail with reference to the drawings.

First Embodiment

FIG. 1 is a sectional view showing an optical glucose sensor chipaccording to a first embodiment.

A glass substrate 1 is provided with a SiO₂ surface layer 2 having athickness of 3 nm or more on its principal surface. A pair of opticalelements, for example, a pair of gratings 3 is formed in the vicinitiesof both ends of the SiO₂ surface layer 2 respectively to introduce lightinto the substrate 1 and to emit the light from the inside of thesubstrate 1. In this case, the optical elements may be substituted witha prism or the like. These gratings 3 are formed of, for example,titanium oxide having a higher refractive index than the above SiO₂surface layer 2. A protective film having a lower refractive index thanthe above gratings 3 may be formed so as to coat the gratings 3. Thisprotective film is made of a material, for example, a fluororesin, inertto a chemical solution and a specimen to be used.

A glucose sensing membrane 4 is formed at a position between thegratings 3 on a part of the surface of the SiO₂ surface layer 2 of thesubstrate 1. This glucose sensing membrane 4 includes a color reagentsubstrate, a first enzyme that oxidizes or reduces glucose, a secondenzyme that reacts with a product obtained by oxidizing or reducingglucose to generate a material making the color reagent substrateexhibit color, a nonionic cellulose derivative, and an ionic polymerinto which a buffer is incorporated. In the glucose sensing membrane 4,at least one of the above first and second enzymes is coated with theionic polymer into which the buffer is incorporated. The above colorreagent substrate, first and second enzymes, ionic polymer and bufferare supported by the above nonionic cellulose derivative.

Here, the standard for coating at least one of the first and secondenzymes with the ionic polymer into which the buffer is incorporated, isdetermined according to the degree of deterioration with time asexplained below.

Specifically, a product obtained by adding glucose to the first enzymeto react is made to act on a specified color reagent substrate, therebycausing the color reagent substrate exhibit color to measure theabsorbance at this time. Then, after this first enzyme is exposed to anatmosphere of a fixed temperature and a fixed humidity for a fixed time,a product obtained by adding glucose to the first enzyme to react ismade to act on a specified color reagent substrate, thereby causing thecolor reagent substrate exhibit color to measure the absorbance at thistime. The ratio of absorbance reduction between the former and thelatter is calculated.

Also, the first and second enzymes are added to glucose and a materialproduced by a reaction with a product of the first enzyme is made to acton a specified color reagent substrate to cause the color reagentsubstrate to exhibit color, thereby measuring the absorbance at thistime. After the second enzyme is exposed to an atmosphere of a fixedtemperature and a fixed humidity for a fixed time in the same manner asin the case of measuring the absorbance of the first enzyme, this secondenzyme is added to glucose together with the first enzyme and a materialproduced by a reaction with a product of the first enzyme is made to acton a specified color reagent substrate to cause the color reagentsubstrate to exhibit color, thereby measuring the absorbance at thistime. The ratio of absorbance reduction between the former and thelatter is calculated.

The absorbance reduction ratio due to the deterioration of the firstenzyme with time is compared with the absorbance reduction ratio due tothe deterioration of the second enzyme with time, to select the onehaving the larger absorbance reduction ratio, and the selected one iscoated with the ionic polymer into which the buffer is incorporated.Also, when the absolute value of the absorbance reduction ratio is largein either the first or second enzyme, it is preferable to coat both withthe ionic polymer into which the buffer is incorporated.

The enzyme and color reagent substrate in the glucose sensing membrane 4are used in the combinations shown in the following Table 1.

TABLE 1 First enzyme Second enzyme Color reagent substrate OxidizingGlucose Peroxidase 3,3′,5,5′-tetramethylbenzidine enzyme oxidaseN,N′-bis(2-hydroxy-3-sulfopropyl)tolidine 3,3′-diaminodenzidineHexokinase Glucose-6- 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-phosphoric acid 2H-tetrazolium bromide dehydrogenase2-(4-rhodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium3-3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]bis(2,5-diphenyl)-2H-tetrazolium choloride Reducing GlucosePhosphorus Aminobenzoic acid enzyme dehydrogenase molybdate

The nonionic cellulose derivative used in the glucose sensing membrane 4is a high-molecular compound participating in the formation of a film.Examples of the nonionic cellulose derivative may include alkylcelluloses such as methyl cellulose and ethyl cellulose; hydroxyalkylcelluloses such as hydroxyethyl cellulose and hydroxypropyl cellulose;hydroxyalkylalkyl celluloses such as hydroxypropylmethyl cellulose,hydroxypropylethyl cellulose, hydroxydiethyl cellulose andhydroxyethylmethyl cellulose; and microfibrous celluloses. Thesematerials may be used either singly or in the form of a mixture.

The above ionic polymer has the function of limiting the precipitationof a salt from the above first and second enzymes during long-termstorage and use. This ionic polymer includes a positive ionic polymerand a negative ionic polymer. Examples of the positive ionic polymerinclude polymers containing a cationic group such as an amino group,guanidino group or biguanide group. Specific examples of the positiveionic polymer include polyallylamine hydrochloride, polyvinylpyridineand polylysine. Examples of the negative ionic polymer include polymerscontaining an anionic group such as phosphates, carboxylates orsulfonates. Specific examples of the negative ionic polymer includepolystyrenesulfonic acid, polyvinylsulfuric acid, polyaspartic acid,polyacrylic acid, polymethacrylic acid, polymaleic acid, polyfumaricacid or cellulose derivatives such as carboxymethyl cellulose andcellulose acetate. Among these ionic polymers, negative ionic polymersare preferable.

The above buffer has the function of controlling the pH and ionicstrength of the first and second enzymes to suppress variations in theforms and structures of these enzymes during long-term storage and use.As this buffer, for example, a phosphoric acid buffer, acetic acidbuffer, citric acid buffer, boric acid buffer, tartaric acid buffer,trishydrochloric acid buffer or carbonic acid buffer may be used.

At least one of the first and second enzymes is coated with the ionicpolymer having incorporated therein such a buffer, whereby not only theprecipitation of a salt from the enzyme during long-term storage and usebut also variations in the forms and structures of these enzymes arelimited to keep these enzymes in highly activated condition.

The above glucose sensing membrane 4 is permitted to contain acrosslinking high-molecular compound. Examples of the crosslinkinghigh-molecular compound may include copolymers of a hydrophilic monomerhaving at least one group selected from a hydroxyl group, carboxylgroup, amino group and ionic functional groups and a hydrophobicmonomer. The copolymer of a hydrophilic monomer and a hydrophobicmonomer is preferably a copolymer of2-methacryloyloxyethylphosphorylcholine and butylacrylate.

The above crosslinking high-molecular compound is preferably containedin the above glucose sensing membrane in an amount of 10⁻⁴ to 10% byweight with respect to all the components of the glucose sensingmembrane. When the content of the crosslinking high-molecular compoundis less than 10⁻⁴% by weight with respect to all the components of theglucose sensing membrane, it is difficult to prevent the phenomena thatthe structure of the film is dissolved and broken under heatingcondition and that the color reagent substrate and enzymes retained invoids in the structure of the film are eluted in an external medium.When the content of the crosslinking high-molecular compound exceeds 10%by weight, on the other hand, there is a fear that the amounts of thecolor reagent substrate and enzymes in the glucose sensing membrane arerelatively reduced and therefore, the sensitivity of the chip islowered.

The above glucose sensing membrane 4 is permitted to further containpolyethylene glycol or ethylene glycol that provides water permeabilityin voids in the structure of the film. The glucose sensing membrane 4containing such polyethylene glycol is increased in hydrophilicproperties and it is therefore possible to raise reaction sensitivitywhen water is used as the medium for introducing glucose.

Next, the action of the optical glucose sensor chip shown in FIG. 1 willbe explained.

An adapter (not shown) having a through-hole (well) is brought intocontact with a specimen, for example, the skin of a human body and theaforementioned sensor chip is attached to the adapter in such a mannerthat the glucose sensing membrane 4 is positioned on the well side. Thisadapter avoids the direct contact of the glucose sensing membrane 4 withthe specimen to contribute to the promotion of the reproducibility ofthe sensing. An extracting medium (for example, a liquid such as wateror physiological brine, which does not react directly with the specimenor sensing membrane but has an affinity to them) is filled in the wellof the adapter on the side on which the glucose sensing membrane ispositioned. Glucose in a skin tissue solution is extracted to theextracting medium from the skin by applying microvoltage to the specimenfrom the outside and further penetrates the sensing membrane 4 from theextracting medium. When the combination of the first and second enzymes(oxidizing or reducing enzyme) and the color reagent substrateconstituting the glucose sensing membrane 4 is, for example, acombination of glucose oxidase (GOD), peroxidase (POD) and3,3′,5,5′-tetramethyl benzidine (TMBZ) shown in the above Table 1, theglucose that has penetrated the sensing membrane 4 is decomposed by GODto generate hydrogen peroxide, which is then decomposed by POD to emitactive oxygen which causes TMBZ to exhibit color. In other words, thechromaticity of TMBZ varies according to the amount of glucose.

In this situation, laser light is made to be incident to the backside ofthe substrate 1 from the laser light source 5 (for example, a laserdiode) through a polar screen (not shown). The incident laser lightpropagates in the substrate 1 including the SiO₂ surface layer 2 whileit is refracted at an interface between the SiO₂ surface layer 2 of thesubstrate 1 and the grating 3 on the left and also at an interfacebetween the SiO₂ surface layer 2 and the glucose sensing membrane 4containing the color reagent substrate which has exhibited color. Atthis time, the evanescent wave of the propagated light is absorbedaccording to the chromaticity correlated with the amount of glucosecontained in the glucose sensing membrane 4. The light propagated in thesubstrate 1 is emitted from the grating 3 on the right and is receivedby a light receiving element 6 (for example, a photodiode). Theintensity of the received laser light is lower than the intensity(initial intensity) of the laser light received when the color of thesensing membrane 4 is not exhibited. As a result, it is possible todetect the amount of glucose from the ratio of intensity reduction ofthe laser light.

Next, explanations will be given of a method of manufacturing theoptical glucose sensor chip shown in FIG. 1.

First, a coating solution for formation of a glucose sensing membrane isprepared by the following method.

(1) At least one of a first enzyme that oxidizes or reduces glucose anda second enzyme that generates a material which makes a color reagentsubstrate exhibit color by a reaction with a product obtained byoxidation or reduction of glucose is mixed in advance with an aqueoussolution containing an ionic polymer and a buffer. In this mixingprocess, the above one of the first and second enzymes is coated withthe ionic polymer into which the buffer is incorporated. In succession,the mixed solution is added to and mixed with the other enzyme, a colorreagent substrate and a nonionic cellulose derivative to prepare aglucose sensing membrane-forming coating solution in which the above oneenzyme coated with the ionic polymer is dispersed together with theother enzyme and color reagent substrate in the nonionic cellulosederivative that is a film forming high-molecular compound.

(2) The aforementioned first and second enzymes are respectively mixedin advance in an aqueous solution containing an ionic polymer and abuffer. In this mixing process, the first and second enzymes are coatedwith the ionic polymer into which the buffer is incorporated. Insuccession, the prepared two mixed solutions are added to and mixed witha color reagent substrate and a nonionic cellulose derivative to preparea glucose sensing membrane-forming coating solution in which the abovefirst and second enzymes respectively coated with the ionic polymer aredispersed together with the above color reagent substrate in thenonionic cellulose derivative that is a film forming high-molecularcompound.

(3) Both the aforementioned first and second enzymes are mixed inadvance in an aqueous solution containing an ionic polymer and a buffer.In this mixing process, the first and second enzymes are coated with theionic polymer into which the buffer is incorporated. In succession, theprepared mixed solution is added to and mixed with a color reagentsubstrate and a nonionic cellulose derivative to prepare a glucosesensing membrane-forming coating solution in which the above first andsecond enzymes coated with the ionic polymer are dispersed together withthe above color reagent substrate in the nonionic cellulose derivativethat is a film forming high-molecular compound.

Then, a pair of optical elements, for example, gratings, is formed onthe substrate. The pair of gratings is formed by the formation of atitanium oxide film on the substrate and by patterning. Successively,the foregoing glucose sensing membrane-forming coating solution isapplied to a substrate area positioned between the gratings, followed bydrying to form a glucose sensing membrane, thereby manufacturing anoptical glucose sensor chip.

As mentioned above, when detecting the amount of glucose by using theoptical glucose sensor chip of the first embodiment, at least one of thefirst and second enzymes in the glucose sensing membrane is coated withthe ionic polymer into which the buffer is incorporated. For thisreason, during long-term storage and use, the precipitation of a saltfrom the enzyme can be suppressed and variations in the shape andstructure of the enzyme are suppressed to keep the enzyme in highlyactivated conditions. As a result, it is possible to provide an opticalglucose sensor chip which can detect glucose amount in a specimen highlysensitively and stably for a long time.

Also, according to the method of the first embodiment, a glucose sensingmembrane-forming coating solution can be prepared in which at least oneof the first and second enzymes is coated with the ionic polymer intowhich the buffer is incorporated and dispersed together with the colorreagent substrate in the nonionic cellulose derivative that is a filmforming high-molecular compound, by using any of the following methods:(a) at least one of the first enzyme that oxidizes or reduces glucoseand the second enzyme that generates a material which makes the colorreagent substrate develop color by a reaction with a product obtained byoxidation or reduction of glucose is mixed in advance with an aqueoussolution containing the ionic polymer and the buffer, and the mixedsolution is added to and mixed with the other enzyme, the color reagentsubstrate and the nonionic cellulose derivative; (b) the aforementionedfirst and second enzymes are respectively mixed in advance with anaqueous solution containing the ionic polymer and the buffer, and therespective prepared mixed solutions are added to and mixed with thecolor reagent substrate and the nonionic cellulose derivative; or (c)both the aforementioned first and second enzymes are mixed in advancewith an aqueous solution containing the ionic polymer and the buffer,and the prepared mixed solution is added to and mixed with the colorreagent substrate and the nonionic cellulose derivative. Thereafter, apair of gratings is formed on the substrate and the foregoing glucosesensing membrane-forming coating solution is applied to a substrate areapositioned between the gratings, followed by drying to form a glucosesensing membrane, thereby manufacturing an optical glucose sensor chipwhich can detect glucose amount in a specimen highly sensitively andstably for a long time.

Second Embodiment

FIG. 2 is a sectional view showing an optical glucose sensor chipaccording to a second embodiment.

A pair of gratings 12 which are optical elements is formed on theprincipal surface of a glass substrate 11 in the vicinities of both endsof the substrate 11 to introduce light into the substrate 11 and to emitthe light from the substrate 11, respectively. These gratings 12 aremade of, for example, titanium oxide having a higher refractive indexthan the above substrate 11. A light-reflecting path layer 13 formed ofa heatcurable or photocurable resin having a higher refractive indexthan the substrate 11 is formed on the principal surface of thesubstrate 11 including the gratings 12. The principal surface of thelight-reflecting path layer 13 is formed in parallel to the principalsurface of the substrate 11 including the gratings 12.

A glucose sensing membrane 14 is formed on the part above thelight-reflecting path layer 13 positioned between the gratings 12. Thisglucose sensing membrane 14 has the same structure as that of the firstembodiment, that is, the structure in which at least one of a firstenzyme that oxidizes or reduces glucose and a second enzyme thatgenerates a material which makes a color reagent substrate exhibit colorby a reaction with a product obtained by oxidation or reduction ofglucose is coated with an ionic polymer into which a buffer isincorporated, and these enzymes, ionic polymer, buffer and color reagentsubstrate are supported by the nonionic cellulose derivative.

Here, the standard for coating at least one of the first and secondenzymes with the ionic polymer into which the buffer is incorporated, isthe same as that explained in the above first embodiment.

The above light-reflecting path layer 13 preferably has a smooth surfaceand a thickness of 10 μm or more and more preferably 10 to 200 μm. Alight-reflecting path layer having a thickness of 10 μm or more makes itpossible to limit a decay of light intensity when light is propagatedand to use, for example, a LED light source in addition to a laserlight.

The first and second enzymes and color reagent substrate in the aboveglucose sensing membrane 14 are used in the combinations shown in theabove Table 1.

As the ionic polymer, buffer and nonionic cellulose derivative in theglucose sensing membrane 14, the same ones exemplified in the abovefirst embodiment may be used.

The above glucose sensing membrane 14 may further contain a crosslinkinghigh-molecular compound and may also contain polyethylene glycol orethylene glycol as explained in the above first embodiment.

Next, the action of the optical glucose sensor chip shown in FIG. 2 willbe explained.

An adapter (not shown) having a through-hole (well) is brought intocontact with a specimen, for example, the skin of a human body and theaforementioned sensor chip is attached to the adapter in such a mannerthat the glucose sensing membrane 14 is positioned on the well side. Anextracting medium including water is filled in the well of the adapteron the side on which the glucose sensing membrane is positioned. Glucosein a skin tissue solution is extracted to the extracting medium from theskin by applying microvoltage to the specimen from the outside andfurther penetrates the sensing membrane 14. When the combination of thefirst and second enzymes (oxidizing or reducing enzyme) and the colorreagent substrate constituting the glucose sensing membrane 14 is, forexample, a combination of glucose oxidase (GOD), peroxidase (POD) and3,3′,5,5′-tetramethyl benzidine (TMBZ) shown in the above Table 1, theglucose that has penetrated the sensing membrane 14 is decomposed by GODto generate hydrogen peroxide, which is then decomposed by POD to emitactive oxygen which causes TMBZ to exhibit color. In other words, thechromaticity of TMBZ varies according to the amount of glucose.

In this situation, laser light is made to be incident to the backside ofthe substrate 11 from the laser light source 15 (for example, a laserdiode) through a polar screen (not shown). The laser light passesthrough the substrate 11 and is refracted at an interface between theprincipal surface of the substrate 11 and the grating 12 on the left,whereby the light is incident to an optical waveguide layer 13. Thelight is also refracted at an interface between the optical waveguidelayer 13 and the glucose sensing membrane 14 containing the colorreagent substrate which has exhibited color to propagate in the opticalwaveguide layer 13. At this time, the evanescent wave of the propagatedlight is absorbed according to the chromaticity correlated with theamount of glucose contained in the glucose sensing membrane 14. Thelight propagated in the optical waveguide layer 13 is emitted from thegrating 12 on the right and is received by a light receiving element 16(for example, a photodiode). The intensity of the received laser lightis lower than the intensity (initial intensity) of the laser lightreceived when the color of the sensing membrane 14 is not exhibited. Asa result, it is possible to detect the amount of glucose from the ratioof intensity reduction of the laser light.

Next, explanations will be given of a method of manufacturing theoptical glucose sensor chip shown in FIG. 2.

First, by using any of the same three methods that are explained in thefirst embodiment, a coating solution for formation of a glucose sensingmembrane is prepared in which at least one of the first and secondenzymes is coated in advance with the ionic polymer into which thebuffer is incorporated and dispersed together with the color reagentsubstrate in the nonionic cellulose derivative that is a film forminghigh-molecular compound.

Then, a pair of optical elements, for example, gratings is formed on theprincipal surface of the glass substrate. The pair of gratings is formedby the formation of a titanium oxide film on the glass substrate and bypatterning. Successively, a light-reflecting path layer made of aheatcurable or photocurable resin having a higher refractive index thanthe substrate is formed on the principal surface of the substrate on theside formed with the grating. Then, the foregoing glucose sensingmembrane-forming coating solution is applied to the part of thelight-reflecting path layer positioned between the gratings, followed bydrying to form a glucose sensing membrane, thereby manufacturing anoptical glucose sensor chip.

As mentioned above, when detecting the amount of glucose by using theoptical glucose sensor chip of the second embodiment, at least one ofthe first and second enzymes in the glucose sensing membrane is coatedwith the ionic polymer into which the buffer is incorporated in the samemanner as in the first embodiment. For this reason, during long-termstorage and use, the precipitation of a salt from the enzyme can besuppressed and variations in the shape and structure of the enzyme aresuppressed to keep the enzyme in highly activated conditions. As aresult, it is possible to provide an optical glucose sensor chip whichcan detect glucose amount in a specimen highly sensitively and stablyfor a long time.

Also, according to the second embodiment, a glucose sensingmembrane-forming coating solution can be prepared in which at least oneof the first and second enzymes is coated with the ionic polymer intowhich the buffer is incorporated and dispersed together with the colorreagent substrate in the nonionic cellulose derivative that is a filmforming high-molecular compound. Thereafter, a pair of gratings isformed on the principal surface of the glass substrate, alight-reflecting path layer having a higher refractive index than thesubstrate is formed on the principal surface of the substrate on theside formed with the gratings, and the foregoing glucose sensingmembrane-forming coating solution is applied to between the gratingsabove the light-reflecting path layer, followed by drying to form aglucose sensing membrane, thereby manufacturing an optical glucosesensor chip which can detect glucose amount in a specimen highlysensitively and stably for a long time.

The present invention will be explained by way of examples.

EXAMPLE 1

Nine μL of a mixture solution of a 0.67 mg/mL peroxidase (POD) solution(dissolved in a 0.01 mol/L phosphoric acid buffer solution [pH: 6.0])and a 5.33 mg/mL glucose oxidase (GOD) solution (dissolved in a 0.01mol/L phosphoric acid buffer solution [pH: 6.0]) was mixed in 1 μL of anaqueous 1 wt % carboxymethyl cellulose (CMC) (negative ionic polymer)solution and the mixture was stirred. Nine μL of the obtained mixturesolution was added to 143.6 μL of isopropyl alcohol (IPA), 116.6 μL ofpurified water, 6 μL of an isopropyl alcohol solution containing 1% byvolume of polyethylene glycol (PEG), 60 μL of an isopropyl alcoholsolution containing 1 mg/mL of 3,3′,5,5′-tetramethylbenzidine (TMBZ), 64μL of an aqueous 2 wt % hydroxyethyl cellulose (HEC) solution and 0.8 μLof an aqueous crosslinking high-molecular compound(2-methacryloyloxyethylphosphorylcholine/butylmethacrylate copolymer)solution and these components were mixed and stirred to prepare 400 μLof a glucose sensing membrane-forming coating solution.

Next, a non-alkali glass substrate which was provided with a SiO₂surface layer having a thickness of 10 nm on its principal surface andhad a refractive index of 1.52 was prepared. A titanium oxide filmhaving a thickness of 50 nm and a refractive index of 2.2 to 2.4 wasformed on the SiO₂ surface layer of this substrate by sputtering. Insuccession, a resist was applied to the titanium oxide film and dried,and then a resist pattern was formed by lithography. Then, by using theresist pattern as a mask, the titanium oxide film was selectivelyremoved by reactive ion etching (RIE), thereby forming gratings on theSiO₂ surface layer in the vicinity of each end of the surface. Then, theresist pattern was removed by ashing.

Next, the above substrate was dry-cleaned by oxygen RIE and then cutinto a chip form having a size of 17 mm×6.5 mm. Then, 8 μL of the aboveglucose sensing membrane-forming coating solution was dripped on thesurface of a sensing membrane forming area positioned between thegratings of the substrate, and dried by purging using inert gas andvacuum drying, thereby forming a porous (water permeable) glucosesensing membrane 0.8 μm in thickness. In this manner, an optical glucosesensor chip shown in the above FIG. 1 was manufactured. A liquid dropletof the glucose sensing membrane-forming coating solution to be drippedhad the following composition.

-   -   Phosphoric acid buffer solution: 0.000525 mol/L    -   Phosphoric acid buffer solution: 0.0003 mol/L    -   PEG: 0.15% by volume    -   TMBZ: 0.15 mg/dL    -   POD: 0.0015 mg/mL    -   GOD: 0.012 mg/mL    -   CMC (negative ionic polymer): 0.0005% by weight    -   HEC: 0.64% by weight    -   2-Methacryloyloxyethylphosphorylcholine and butylmethacrylate        copolymer: 0.002% by weight

EXAMPLE 2

An optical glucose sensor chip shown in the above FIG. 1 was produced byforming a sensing membrane in the same manner as in Example 1 exceptthat polylysine as a positive ionic polymer was blended in the dropletof the glucose sensing membrane-forming coating solution in an amount of0.0008 μg/L in place of CMC as a negative ionic polymer in Example 1.

COMPARATIVE EXAMPLE 1

An optical glucose sensor chip shown in the above FIG. 1 was produced byforming a sensing membrane in the same manner as in Example 1 by using aglucose sensing membrane-forming coating solution prepared by one mixingoperation using the same components as in Example 1 except that theionic polymer and the sodium phosphate buffer were not contained.

With regard to the optical glucose sensor chips obtained in Examples 1and 2 and Comparative Example 1, a variation in sensitivity (absorbance)to glucose with time was measured using the following method.

Specifically, an adapter having a through-hole (well) was brought intocontact with a proper flat plate (for example, a glass plate). Eachsensor chip was attached to this adapter in such a manner that theglucose sensing membrane was disposed on the well side to partition thewell. In the situation (temperature: 35° C.) where an aqueous solutioncontaining 1 mg/dL of glucose was filled in the well, laser light wasmade to be incident to the backside of the substrate 1 from a laserdiode 5 through a polar screen as shown in FIG. 1. The incident laserlight was refracted at an interface between the SiO₂ surface layer 2 ofthe substrate 1 and the grating 3 on the left, and also refracted at aninterface between the SiO₂ surface layer 2 and the glucose sensingmembrane 4 containing the color reagent substrate which had exhibitedcolor to propagate in the substrate 1 including the SiO₂ surface layer2. The laser light propagated by the refraction at an interface betweenthe grating 3 on the right and the substrate 1 was received by aphotodiode 6 to measure the intensity (absorbance) of the received laserlight.

The same operations as above were carried out after the sensor chip hadbeen stored for one day, 14 days, 40 days and 100 days in the case ofthe sensor chips of Examples 1 and 2 respectively and after the sensorchip had been stored for one day, 7 days and 90 days in the case of thesensor chip of Comparative Example 1.

The results of these operations are shown in FIG. 3.

As can be understood from FIG. 3, the sensitivity of the sensor chip ofComparative Example 1 deteriorates more after the chip has been storedfor 7 days and more significantly after the chip has been stored for 90days compared with the sensitivity obtained before the sensor is stored.

It can be understood, on the other hand, that each sensor chip ofExamples 1 and 2 has a sensitivity similar to that obtained before thesensor chip is stored even after the sensor chip has been stored for 100days. It is found that, particularly, the sensor chip of Example 1provided with a glucose sensing membrane containing a negative ionicpolymer has a higher capability of maintaining sensitivity with thepassage of storage time than that of Example 2 provided with a glucosesensing membrane containing a positive ionic polymer.

Similarly to Example 1, the glucose sensor chip shown in FIG. 2 providedwith a light-reflecting path layer made of a heatcurable or photocurableresin having a higher refractive index than the substrate could maintainhigh sensitivity even after being stored for a long time.

Also, as each of the first enzyme, second enzyme and color reagentsubstrate supported by the glucose sensing membrane used in the aboveembodiments and examples, only one material is selected. However, pluralmaterials may be combined according to the purpose of use.

Moreover, glass is used as the substrate in the above embodiment.However, no restriction is imposed on this material as long as it hasthe characteristics allowing reference light to propagate and betransmitted. Film bodies of single crystals and various resin materialsmay also be used, such as heatcurable resin materials, thermoplasticresin materials and photocurable resin materials.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

1. An optical glucose sensor chip comprising: a substrate; a pair ofoptical elements formed on a principal surface of the substrate forintroducing light into the substrate and for emitting the light from thesubstrate; and a glucose sensing membrane formed on the principalsurface of the substrate at a position between the optical elements;wherein the sensing membrane includes a color reagent substrate, a firstenzyme which oxidizes or reduces glucose, a second enzyme that generatesa material which makes the color reagent substrate exhibit color by areaction with a product obtained by oxidation or reduction of glucose, anonionic cellulose derivative, and an ionic polymer into which a bufferis incorporated, at least one of the first and second enzymes is coatedwith the ionic polymer, and the color reagent substrate, the first andsecond enzymes, the buffer and the ionic polymer are supported by thenonionic cellulose derivative.
 2. The sensor chip according to claim 1,wherein the first enzyme is glucose oxidase, the second enzyme isperoxidase and the color reagent substrate is at least one of3,3,5,5-tetramethylbenzidine andN,N′-bis(2-hydroxy-3-sulfopropyl)tridine.
 3. The sensor chip accordingto claim 1, wherein the ionic polymer is a negative ionic polymer. 4.The sensor chip according to claim 3, wherein the negative ionic polymeris a polymer containing at least one anionic group selected from thegroup consisting of a phosphate, a carboxylate and a sulfonate.
 5. Thesensor chip according to claim 1, wherein the nonionic cellulosederivative is at least one selected from the group consisting of analkyl cellulose, a hydroxyalkyl cellulose and a hydroxyalkylalkylcellulose.
 6. The sensor chip according to claim 1, wherein the sensingmembrane further contains a crosslinking high-molecular compound.
 7. Thesensor chip according to claim 6, wherein the crosslinkinghigh-molecular compound is a copolymer of a hydrophobic monomer and ahydrophilic monomer having at least one group selected from the groupconsisting of a hydroxyl group, a carboxyl group, an amino group and anionic functional group.
 8. The sensor chip according to claim 7, whereinthe copolymer of a hydrophilic monomer and a hydrophobic monomer is acopolymer of 2-methacryloyloxyethylphosphorylcholine andbutylmethacrylate.
 9. The sensor chip according to claim 1, wherein theglucose sensing membrane further contains polyethylene glycol orethylene glycol for endowing the membrane with water permeability. 10.An optical glucose sensor chip comprising: a glass substrate; a pair ofoptical elements formed on a principal surface of the glass substratefor introducing light into the glass substrate and for emitting thelight from the glass substrate; a light-reflecting path layer formed onthe principal surface of the substrate on which the optical elements areformed and made of a resin having a higher refractive index than thesubstrate; and a glucose sensing membrane formed on the light-reflectingpath layer at a position between the optical elements; wherein thesensing membrane includes a color reagent substrate, a first enzymewhich oxidizes or reduces glucose, a second enzyme that generates amaterial which makes the color reagent substrate exhibit color by areaction with a product obtained by oxidation or reduction of glucose, anonionic cellulose derivative, and an ionic polymer into which a bufferis incorporated, at least one of the first and second enzymes is coatedwith the ionic polymer, and the color reagent substrate, the first andsecond enzymes, the buffer and the ionic polymer are supported by thenonionic cellulose derivative.
 11. The sensor chip according to claim10, wherein the first enzyme is glucose oxidase, the second enzyme isperoxidase and the color reagent substrate is at least one of3,3,5,5-tetramethylbenzidine andN,N′-bis(2-hydroxy-3-sulfopropyl)tridine.
 12. The sensor chip accordingto claim 10, wherein the ionic polymer is a negative ionic polymer. 13.The sensor chip according to claim 12, wherein the negative ionicpolymer is a polymer containing at least one anionic group selected fromthe group consisting of a phosphate, a carboxylate and a sulfonate. 14.The sensor chip according to claim 10, wherein the nonionic cellulosederivative is at least one selected from the group consisting of analkyl cellulose, a hydroxyalkyl cellulose and a hydroxyalkylalkylcellulose.
 15. The sensor chip according to claim 10, wherein thesensing membrane further contains a crosslinking high-molecularcompound.
 16. The sensor chip according to claim 15, wherein thecrosslinking high-molecular compound is a copolymer of a hydrophobicmonomer and a hydrophilic monomer having at least one group selectedfrom the group consisting of a hydroxyl group, a carboxyl group, anamino group and an ionic functional group.
 17. The sensor chip accordingto claim 16, wherein the copolymer of a hydrophilic monomer and ahydrophobic monomer is a copolymer of2-methacryloyloxyethylphosphorylcholine and butylmethacrylate.
 18. Thesensor chip according to claim 10, wherein the glucose sensing membranefurther contains polyethylene glycol or ethylene glycol for endowing themembrane with water permeability.
 19. A method of manufacturing anoptical glucose sensor chip, comprising: preparing a glucose sensingmembrane-forming coating solution by using any of: (a) a method in whichat least one of a first enzyme that oxidizes or reduces glucose and asecond enzyme that generates a material which makes a color reagentsubstrate exhibit color by a reaction with a product obtained byoxidation or reduction of glucose is mixed in advance with an aqueoussolution containing an ionic polymer and a buffer, and the mixedsolution is added to and mixed with the other enzyme, a color reagentsubstrate and a nonionic cellulose derivative; (b) a method in which thefirst and second enzymes are respectively mixed in advance with anaqueous solution containing an ionic polymer and a buffer, and therespective prepared mixed solutions are added to and mixed with a colorreagent substrate and a nonionic cellulose derivative; or (c) a methodin which both the first and second enzymes are mixed in advance with anaqueous solution containing an ionic polymer and a buffer, and theprepared mixed solution is added to and mixed with a color reagentsubstrate and a nonionic cellulose derivative; forming a pair of opticalelements on a substrate for introducing light into the substrate and foremitting the light from the substrate; and applying the glucose sensingmembrane-forming coating solution to a substrate area positioned betweenthe optical elements, followed by drying to form a glucose sensingmembrane.
 20. A method of manufacturing an optical glucose sensor chip,comprising: preparing a glucose sensing membrane-forming coatingsolution by using any of: (a) a method in which at least one of a firstenzyme that oxidizes or reduces glucose and a second enzyme thatgenerates a material which makes a color reagent substrate exhibit colorby a reaction with a product obtained by oxidation or reduction ofglucose is mixed in advance with an aqueous solution containing an ionicpolymer and a buffer, and the mixed solution is added to and mixed withthe other enzyme, a color reagent substrate and a nonionic cellulosederivative; (b) a method in which the first and second enzymes arerespectively mixed in advance with an aqueous solution containing anionic polymer and a buffer, and the respective prepared mixed solutionsare added to and mixed with a color reagent substrate and a nonioniccellulose derivative; or (c) a method in which both the first and secondenzymes are mixed in advance with an aqueous solution containing anionic polymer and a buffer, and the prepared mixed solution is added toand mixed with a color reagent substrate and a nonionic cellulosederivative; forming a pair of gratings on a substrate for introducinglight into the substrate and for emitting the light from the substrate;forming a light-reflecting path layer made of a resin having a higherrefractive index than the substrate on a principal surface of thesubstrate on which the optical elements are formed; and applying theglucose sensing membrane-forming coating solution to the part of thelight-reflecting path layer positioned between the optical elements,followed by drying to form a glucose sensing membrane.